The number of treatment options available to oncologists has dramatically increased in recent years due to major advances in our understanding of the signaling pathways that drive cancer development. A critical cell cycle checkpoint regulator is the p53 tumor suppressor, which is kept in check in normal cells by HdmX and Hdm2. Not surprisingly, p53 is inactivated in most human cancers, either by mutation or overexpression of Hdm2 or HdmX. Given its recently described role in suppressing a cancer stem cell (CSC) transcriptional profile, which is a hallmark of triple-negative breast cancer (TNBC) subtypes, we are proposing a new strategy aimed at reactivating p53 for the benefit of TNBC patients. Using a TNBC model, our preliminary studies have identified high levels of HdmX protein in the CSC population. Currently, DNA-damaging chemotherapy is the first-line therapy for TNBC. However, the side effects from this approach are significant and often dose limiting because DNA damage is unable to activate mutant p53 or sufficiently release wild-type p53 from HdmX. Moreover, an increased proportion of CSC compared to other breast cancer subtypes makes rapid recurrence a significant issue. The most advanced molecules currently in clinical trials for reactivating p53 include the Hdm2 antagonist Nutlin-3 and the mutant p53 reactivating compound PRIMA-1. However, Nutlin-3 specifically disrupts Hdm2-p53 but not HdmX-p53 interactions. Thus, the initial excitement over Nutlin-3 has been dampened by observations that tumors harboring high levels of HdmX are resistant to Nutlin-3 treatment. We propose TNBC CSC will not respond to Nutlin-3, or other Hdm2-specific molecules. Towards identifying HdmX- specific inhibitors to help combat TNBC, we established an assay to detect p53 activation in the presence of high-level HdmX expression and performed a high-throughput screen. We describe here the discovery of a selective inhibitor of HdmX named CTX1. The hypothesis of this proposal is that HdmX is a key regulator of CSC in TNBC, and that combined suppression of HdmX and Hdm2 will efficiently kill TNBC CSC and prevent tumor growth. We have devised two aims for the current study. In AIM 1, we will determine the mechanism and efficacy of CTX1-mediated growth suppression in TNBC. We will test whether suppression of both HdmX and Hdm2 by combined CTX1/Nutlin-3 therapy results in the sustained activation of wild-type p53, as well as mutant p53 reactivated by PRIMA-1. In AIM 2, we will examine the suppression of TNBC stem cells by combination therapy involving CTX1 and Nutlin-3 using xenograft models of CSC generated in vitro and CSC isolated from primary triple-negative breast cancers. In summary, CTX1 is an excellent compound for further studies as it exhibits high anti-cancer activity, high specificity for HdmX, and a synergy with the Hdm2 inhibitor Nutlin-3. Moreover, because TNBC patients have an increased risk of metastatic disease, respond poorly to current chemotherapeutic regimens, have a short time to recurrence, and poor overall survival, they are the ideal candidates who could benefit most from combined p53-reactivation therapy, as described here.